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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 林頌然 | |
| dc.contributor.author | Tsung-Sheng Lee | en |
| dc.contributor.author | 李宗昇 | zh_TW |
| dc.date.accessioned | 2021-06-16T03:43:58Z | - |
| dc.date.available | 2020-02-25 | |
| dc.date.copyright | 2015-02-25 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-02-09 | |
| dc.identifier.citation | [1] Headington JT. Cicatricial alopecia. Dermatologic clinics. 1996;14:773-82.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55001 | - |
| dc.description.abstract | 毛囊在每個生長週期能夠產生新毛髮的一個器官。毛囊的發育、生長和再生週期循環是由毛囊內部的上皮－間質之間廣泛且密集的交互作用支配與協同所產生。因此，以組織工程的方式於體外發展一套能夠模擬三維毛囊類結構的系統，將是研究瞭解毛囊內部上皮－間質交互作用的一大利器，也是本實驗研究最主要目標。
本研究主要目的是開發三維共培養系統，用以能夠高效生產角質－真皮乳突毛囊組織球，模擬仿造毛囊內部上皮－間質訊息溝通與交互作用，以應用於毛囊之研究。本研究可分為三大主軸探討：細胞擴展、製造類毛囊球微組織及功能檢測。 第一、細胞擴展：建立細胞長程培養方法，並於體外大規模地繁殖真皮乳突細胞 (dermal papilla cells) 與角質細胞 (keratinocytes) 以利研究進行。有效及大規模長程培養成鼠角質細胞方法的建立，透過 3T3-J2 cells 的滋養、表皮生長因子 (epidermal growth factor, EGF) 與霍亂毒素 (cholera toxin, CT) 的額外添加促進細胞複製增殖及生長，克服了成鼠角質細胞不易培養的挑戰。長程培養所得的角質細胞不僅細胞型態正常，且為具強力形成細胞群落能力與具分化力的基底層角質細胞型態。 第二、製造類毛囊球微組織：利用角質細胞及真皮乳突細胞於親水性生醫材料不貼附的特性，先將真皮乳突細胞種植於已塗佈聚乙烯醇 (PVA) 的 PCR 孔盤內並快速聚集為單一細胞球體；而後，再植入角質細胞於管內與成球的真皮乳突細胞共培養，藉此形成毛囊球內部的雙層構造（即真皮乳突細胞於中心，角質細胞包覆於外圍）。其中特別的是，細胞存活率於共培養其間內皆達 90% 之上。 第三、功能檢測：藉由測試與毛囊生長發育及分化的重要識別蛋白與基因表現（如 Keratin 6, Keratin 14, Keratin 75, AE 13, LEF1），發現組織球內部保有毛囊內部上皮－間質交互作用訊號溝通特性並誘使組織球趨往毛囊分化的路徑。此外，組織球內兩層細胞彼此間亦如同毛囊結構形成基底膜，且在毛囊再生試驗中證實植入的類毛囊球具有誘導毛囊新生能力而生成髮幹。如此種種特徵證實所製造的類毛囊球組織不論結構、細胞、功能表現皆與真實毛囊球極度相仿。 本實驗成功地建立了簡單的組織真皮乳突細胞與角質細胞之共培養系統，以形成類似毛囊球雙層細胞的排列構造，模擬與促進毛囊內部上皮－間質交互作用，達成體外發展出培養毛囊雛形組織，並於體內具誘導毛囊再生力的目標。此外，此系統更具有搭配自動化設備大量生產的潛力。於未來，此大規模生產類毛囊球系統，不僅有應用於藥物測試的潛力，甚至能推廣至人體其他上皮與間質相互作用方式發育及再生的器官研究。 | zh_TW |
| dc.description.abstract | The hair follicle is a regenerating organ which produces a new hair shaft during each growth cycle. The morphogenesis, development and cycling of the hair follicle is dependent on extensive ,collaborative and well-orchestrated epithelial-mesenchymal interaction. Accordingly, development of an engineered three-dimensional hair follicle organoid model can help to explore and test the epithelial-mesenchymal interaction in vitro.
The aim of this study was to develop a three-dimensional co-culture system for efficient production of folliculoid keratinocyte-dermal papilla (DP) microtissues to imitate epithelial-mesenchymal interaction in vitro. The study consists of three parts: cell expansion, production of hair bulb-like structure and functional testing. The first was to establish methods for cell expansion. We constituted the method for long-term adult C57BL/6 mouse keratinocyte culture using fibroblast feeder layers, EGF and cholera toxin. The keratinocytes obtained were morphologically normal with hight proliferative ability. Secondly, we developed a method for scalable production of hair follicle bulb-like microtissues. Due to the poor adherence of DP cells and keratinocytes to hydrophilic polyvinyl alcohol (PVA), the seeded DP cells in PVA-coated 96-welled commercial PCR tube arrays quickly aggregate into single spheroids with progressive compaction. Keratinoctes were then seeded for co-culturing. DP cells and keratinocytes quickly formed a hybrid spheroid with a core-shell structure with DP cells surrounded by keratinocytes. This structure was similar to the native hair bulb. In our method, cell viability remained as high as 90% in the microtissues. Thirdly, we examined the differentiation and function of the hair bulb-like microtissues. We were able to investigate and identify several markers important for the hair follicle development and differentiation (for example, Keratin 6, Keratin 14, Keratin 75, AE 13, LEF1). We showed that these hair bulb-like spheroids, cultured under well-defined conditions, retained several crucial characters of hair follicle epithelial-mesenchymal interaction. We found that DP cells and keratinocytes were in close physical contact with production basement membrane extracellular matrix components. Functionally, these spheroids could regenerate new hair follicles after transplantation in vivo. We successfully set up an easy system for fast organization of DP cells and keratinocytes into follicular structures, that resembled the native hair bulb orientation with epithelial layer surrounding the DP aggregate, to mimic and promote epithelial-mesenchymal interaction. Furthermore, this method is of scalable potential with automatic equipment. In the future, our system of mass preparation of 3D engineered follicular structures is potential to be not only use in drug testing assays, but also applied to mass generation of other epithelial organ primordia in vitro. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T03:43:58Z (GMT). No. of bitstreams: 1 ntu-104-R01548065-1.pdf: 8050700 bytes, checksum: 579608aea1800feed8599df47642b891 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 碩士論文口試委員會審定書 i
誌謝 ii 摘要 iii Abstract v 1 第一章 緒論 1 1.1 禿髮與治療 1 1.2 毛髮結構 4 1.3 毛囊發育 6 1.4 毛囊再生的發展 8 1.5 三維成球 12 1.6 生醫材料：聚乙烯醇 (polyvinyl alcohol, PVA) 15 2 第二章 研究目標與實驗設計 16 2.1 研究目標 16 2.2 實驗設計 18 3 第三章 實驗材料與方法 20 3.1 C57BL/6 鼷鼠真皮乳突細胞培養 20 3.2 C57BL/6 鼷鼠角質細胞分離 21 3.3 C57BL/6 鼷鼠角質細胞長程培養 22 3.4 高分子材料製備 24 3.5 立體雙層組織球共培養方式 24 3.6 三維組織球型態觀察 25 3.7 Hematoxylin & Eosin 染色 (H&E staining) 25 3.8 組織球體內細胞存活率分析 (cell viability assay) 26 3.9 TUNEL assay (TdT-mediated dUTP Nick-End Labeling assay) 26 3.10 RNA 萃取 27 3.11 逆轉錄聚合酶連鎖反應 (Reverse transcription, RT) 28 3.12 即時定量聚合酶連鎖反應 (Real-Time Quantitative Polymerase Chain Reaction, qPCR) 28 3.13 雙重免疫螢光染色 (Double immunofluorescence stain) 30 3.14 動物實驗 32 4 第四章 實驗結果 33 4.1 C57BL/6 鼷鼠角質細胞二維長程培養細胞型態觀察 33 4.2 建立三維共培養系統製造類毛囊球結構 35 4.2.1 細胞球體結構的觀測 37 4.2.2 細胞球體結構的分析 39 4.3 三維共培養之組織球聚合分析 41 4.4 類毛囊球細胞鑑定 44 4.5 類毛囊球細胞存活觀察及增殖分析 (Cell viability assay, TUNEL assay, Proliferation assay) 46 4.6 探討三維共培養系統是否具細胞比例可調性 49 4.7 探討三維共培養系統是否具細胞種類可調性 53 4.8 探討三維共培養系統內細胞種植順序對成球影響性 58 4.9 類毛囊球之上皮－間質交互作用對於類毛囊球組織分化表現 62 4.9.1 組織球是否具備分化能力的探討 62 4.9.2 組織球是否趨往表皮分化的探討 65 4.9.3 組織球是否趨往毛囊分化的探討 68 4.9.4 組織球內部是否有基底膜成形的探討 80 4.10 毛囊再生能力 82 5 第五章 討論 85 6 第六章 結論 89 7 第七章 參考文獻 90 | |
| dc.language.iso | zh-TW | |
| dc.subject | 聚乙烯醇 | zh_TW |
| dc.subject | 角質細胞 | zh_TW |
| dc.subject | 類毛囊球 | zh_TW |
| dc.subject | 上皮－間質交互作用 | zh_TW |
| dc.subject | 毛囊再生 | zh_TW |
| dc.subject | 三維培養 | zh_TW |
| dc.subject | hair follicle regeneration | en |
| dc.subject | epithelial-mesenchymal interaction | en |
| dc.subject | three dimension culture | en |
| dc.subject | keratinocyte | en |
| dc.subject | polyvinyl alcohol | en |
| dc.subject | hair bulb-like spheroids | en |
| dc.title | 於聚乙烯醇表面建立三維共培養系統製造類毛囊球結構應用於上皮－間質交互作用之探討 | zh_TW |
| dc.title | Establishment of three-dimensional co-culture system on polyvinyl alcohol surface to produce hair bulb-like microtissues: a model for exploring epithelial-mesenchymal interaction | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊台鴻,陳文彬 | |
| dc.subject.keyword | 上皮－間質交互作用,三維培養,角質細胞,聚乙烯醇,類毛囊球,毛囊再生, | zh_TW |
| dc.subject.keyword | epithelial-mesenchymal interaction,three dimension culture,keratinocyte,polyvinyl alcohol,hair bulb-like spheroids,hair follicle regeneration, | en |
| dc.relation.page | 98 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-02-10 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
| Appears in Collections: | 醫學工程學研究所 | |
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|---|---|---|---|
| ntu-104-1.pdf Restricted Access | 7.86 MB | Adobe PDF |
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